Choline is a dietary essential nutrient that is required for the synthesis of membrane phospholipids, betaine, osmolytes and the neurotransmitter acetylcholine. To carry out these metabolic roles, choline is delivered into cells by at least two transport mechanisms. One mechanism is a sodium-dependent high-affinity mechanism found only in acetylcholine-containing neurons. The second mechanism is a sodium-independent, low-affinity transport (LACT) that is present in all cells. Little is known about the LACT protein or even whether there might be multiple LACT proteins. Biochemical studies by others and confirmed by the present laboratory have shown that LACT is a type of organic cation transporter (OCT). However, the substrate specificity of LACT does not match that of known OCT proteins. By analysis of the OCT gene family, we identified several uncharacterized genes closely related to known OCT proteins. We hypothesize that the LACT gene is one of these orphan genes. The experiments in this proposal will test this hypothesis and consist of 2 specific aims, one genetic and one biochemical. 1. Expression of orphan OCT genes in the NG108 cell line, will be eliminated by transfection with siRNA oligonucleotides. Transport of choline will be measured in mRNA-silenced cells to determine if LACT activity is also reduced. 2. Orphan OCT genes will be expressed in yeast Saccharomyces cerevisiae and tested for choline transport activity. These two specific aims are complementary approaches that will test, decisively, the hypothesis. Should the orphan genes not display LACT activity, yeast expressing the novel proteins will be screened for uptake of candidate substrates in order to identify the physiological substrate(s) for the orphan genes. RNA silencing will then be done to confirm the role of the orphan transporter in transport of identified substrates. These experiments will provide a better understanding of this important gene family and insight into delivery of choline to cells. Such insights would provide avenues to reverse the loss of membrane integrity and impaired cellular function in disorders such as stroke. Analysis of OCT protein function will also provide new information on the uptake, excretion and distribution of cationic drugs. [unreadable] [unreadable] [unreadable]